L-carnitine plays a fundamental role in metabolism, being a key element in the oxidation of long-chain fatty acids and thus in the production of energy.
There are many pathological states the underlying cause of which is a deficiency of L-carnitine, and L-carnitine determination is necessary in order to establish the precise a etiology of the related disease processes.
There has been a growing awareness among clinicians over recent years of the importance of L-carnitine deficiencies (Bieber et is al. Fed. Proc. 41, 2858 (1982); Stanley, Adv. Pediatr. 34, 59 (1987).
One of the first methods used for determining L-carnitine (hereinafter called carnitine for short) was described by Marquis and Fritz in 1964 (Journal of Lipid Research 5, 184-187). Various other methods have been described, such as, for example, those by Marzo et al., J. Chromatogr. 527, 247 (1990); and Hoppel, in: Hommes (ed.), Techniques in Diagnostic Human Biochemical Genetics, New York, Wiley-Liss, 309-326 (1991).
The most widespread methods are based on the reaction of the enzyme carnitine acetyl transferase (CAT; EC 2.3.1.7): 
One very widespread method for the indirect determination of carnitine is based on the reaction of acetyl-Coenzyme A, released by the preceding reaction with 5,5′-dithiobis-2-nitrobenzoate (DTNB), which in turn releases the thiophenylate ion, which is determined spectrophotometrically at 412 nm (see, in addition to the above-mentioned Marquis, Seccombe D. W., Clinical Chemistry, 22, No. 10, 1589-1592, 1976; Pearson, Methods of Enzymatic Analysis, 4, 1758-1771, 2nd edition—Bergmeyer; Casillas E. R. Biochimica et Biophysica Acta, 184, 566-577, 1969; Cederblad G. Clinica Chimica Acta, 33, 117.123, 1971; Carrier H. N., Muscle & Nerve July/August 326-328, 1980). This method determines free carnitine. For the determination of the short-chain acyl carnitines, and thus of total carnitines, the sample is subjected to alkaline hydrolysis, converting the acyl carnitines to free carnitine.
The known methods entail elaborate preparation phases, or use techniques which are only poorly conducive to automation of the analyses. For a discussion of the subject, see U.S. Pat. No. 5,316,917, filed in the name of Duke University, and incorporated herein for reference purposes. This patent aims to solve the problem of the automation of the analyses. This need is strongly felt in clinical laboratories that have to carry out large numbers of determinations. The Duke University patent offers the solution of an automated spectrophotometric method, consisting in the following steps:    1) addition of a plurality of samples of deproteinized biological fluids to a set of wells of a centrifugal spectrophotometric analyzer;    2) addition to each sample of:            a) acetyl-Coenzyme A in an amount sufficient to react essentially with all the free carnitine in the sample to produce acetyl-carnitine and free Coenzyme A;        b) DTNB in an amount sufficient to convert essentially all the Coenzyme A produced in the previous reaction to thiophenylate, and then        c) simultaneously combine CAT, in each sample, in an amount sufficient to set off the reaction between acetyl-Coenzyme A and free carnitine and bring it to completion, and then        d) determine the amount of thiophenylate present in the sample simultaneously by spectrophotometry.        The kit used to implement the method described in the patent cited includes:                    a) a first container containing a solution of DTNB at a concentration ranging from 0.27 to 27 mmol/L, at a pH ranging from 6.5 to 8.5, and            b) a second container containing a solution consisting of acetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L, where the two solutions can be mixed together prior to use to form a solution containing DTNB at a concentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17 mmol/l.                        
In actual fact, the kit must also include a third container containing from 4 to 40 kU/L of CAT, for example in the form of a solution at a concentration ranging from 1.72 to 172 kU/L. It is envisaged that the third container should contain CAT in lyophilized form. Also envisaged in the kit are containers with carnitine standards in aqueous solution from 0.1 to 10 mmol/L and of octanoyl L-carnitine from 0.1 to 10 mmol/L. Also provided in the kit is a fourth container containing a 3-[N-morpholine]propanesulphonic acid hydrochloric solution, from 0.1 to 10 mmol/L.
The execution of the procedure entails the preparation of 4 solutions:                1. DTNB; HEPES; EDTA at pH 7.5        2. MOPS-HCl        3. KOH        
In fact, the procedure described in the patent is called “three reagent chemistry”.
It is known that the solutions envisaged in the kit described above must be stored at low temperature, from 0 to 4° C., as prescribed in the patent cited.
U.S. Pat. No. 6,027,690, a divisional patent of U.S. Pat. No. 5,872,008, filed in the name of Bair and Shug, provides a kit for the diagnosis of premenstrual syndrome, based on the determination of free and total carnitine in the blood. Apart from the specific indication of the method, to implement the method the kit consists of:                a) a first container containing a solution of acetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L, and        b) a second container containing a solution consisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from 0.27 to 27 mmol/L at a pH ranging from 6.5 to 8.5, where the two solutions can be mixed together prior to use to form a solution consisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17 mmol/l.        
In the execution of the procedure according to U.S. Pat. No. 6,072,690, the method described in U.S. Pat. No. 5,316,917 can be adopted amongst others.
A Boehringer Mannheim kit is commercially available for the determination of L-carnitine in plasma, seminal fluid, and urine. The determination is based on the following reactions: The amount of NADH consumed during the reaction is equivalent to half the amount of L-carnitine. NADH is determined by absorbance at 334 (Hg), 340 or 365 (Hg) nm.
The kit comprises:                1) 3 vials (1) each containing 0.7 g of a Coenzyme/buffer mixture consisting of Tris buffer pH 7.0, NADH 5 mg, ATP 6 mg, acetyl-Coenzyme A 4 mg, PEP 3 mg, magnesium acetate, and stabilizing agents.        2) Vial (2) containing approximately 3 ml of enzyme suspension consisting of acetyl-CoA synthetase (ACS), 2U approx., myokinase (MK), 160 U approx., lactate dehydrogenase (LDH), 240 U approx., and pyruvate kinase (PK), 240 U approx.        3) Vial (3) containing 0.2 ml of carnitine acetyl transferase (CAT) enzyme suspension, 60 U approx.        4) Vial (4) containing L-carnitine standard.        5) Vial (5) containing detergent solution.        
Prior to use, the contents of one vial (1) are diluted with 10 ml of distilled water and 1 ml is added from vial (5). The other vials are used as such. The procedure consists in the addition of the sample (or standard) to solution (1), then addition of suspension (2), measurement of absorbance, then addition of suspension (3) and subsequent measurement of absorbance.